Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor.

Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY-induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.

Lysophosphatidic acid is detectable in human bronchoalveolar lavage fluids at baseline and increased after segmental allergen challenge.

BACKGROUND: Lysophosphatidic acid (LPA) is a biologically active lysophospholipid and a component of normal plasma. LPA binds to receptors expressed on circulating and structural lung cells and affects cell growth and activation. Whether LPA is present in the lung has not been previously reported. OBJECTIVE: To develop an assay to measure LPA in bronchoalveolar lavage (BAL) fluids, and to study the association between LPA and allergic airway inflammation. METHODS: Seventeen allergic subjects underwent bronchoscopy and segmental allergen challenge, followed 18 h later by BAL. Supernatants were analysed for LPA content using liquid chromatography and mass spectroscopy. Expression of LPA receptors on primary bronchial epithelial cells was analysed by immunolabelling, and the effects of LPA on epithelial cell barrier function was investigated by measuring transepithelial resistance. RESULTS: LPA was detectable in BAL from control lung segments, and significantly increased 18 h after allergen challenge. Polyunsaturated species of LPA were especially increased following segmental allergen challenge. LPA levels did not strongly correlate with the number or percentages of eosinophils, neutrophils of lymphocytes, whereas MIP-3alpha (CCL20) levels correlated significantly with the allergen-driven influx of lymphocytes. The levels of LPA from control sites correlated inversely with BAL protein content, suggesting that LPA promoted epithelial barrier integrity at baseline. Experiments using primary human bronchial epithelial cells confirmed that LPA tightened the epithelial cell barrier. CONCLUSION: Lysophosphatidic acid is detectable in human BAL fluids at baseline and its expression increases during allergic inflammation. LPA does not appear to be a dominant chemoattractant for eosinophils or lymphocytes during allergic airway inflammation. In the absence of ongoing inflammation, LPA may promote epithelial barrier integrity.

Diverse effects of vascular endothelial growth factor on human pulmonary endothelial barrier and migration.

Increased endothelial permeability is involved in the pathogenesis of many cardiovascular and pulmonary diseases. Vascular endothelial growth factor (VEGF) is a permeability-increasing cytokine. At the same time, VEGF is known to have a beneficial effect on endothelial cells (EC), increasing their survival. Pulmonary endothelium, particularly, may be exposed to higher VEGF concentrations, since the VEGF level is the higher in the lungs than in any other organ. The purpose of this work was to evaluate the effects of VEGF on barrier function and motility of cultured human pulmonary EC. Using transendothelial resistance measurements as an indicator of permeability, we found that 10 ng/ml VEGF significantly improved barrier properties of cultured human pulmonary artery EC (118.6+/-0.6% compared with 100% control, P<0.001). In contrast, challenge with 100 ng/ml VEGF decreased endothelial barrier (71.6+/-1.0% compared with 100% control, P<0.001) and caused disruption of adherens junctions. VEGF at both concentrations increased cellular migration; however, 10 ng/ml VEGF had a significantly stronger effect. VEGF caused a dose-dependent increase in intracellular Ca2+ concentration; however, phosphorylation of myosin light chain was detectably elevated only after treatment with 100 ng/ml. In contrast, 10 ng/ml but not 100 ng/ml VEGF caused a significant increase in intracellular cAMP (known barrier-protective stimulus) compared with nonstimulated cells (1,096+/-157 and 610+/-86 fmol/mg, respectively; P<0.024). Y576-specific phosphorylation of focal adhesion kinase was also stimulated by 10 ng/ml VEGF. Our data suggest that, depending on its concentration, VEGF may cause diverse effects on pulmonary endothelial permeability via different signaling pathways.